Introduction
Tea is the second highest consumed nonalcoholic beverage worldwide, and an important dietary source of flavonoid compounds [38]. Although these tea polyphenols possess therapeutic properties, including anti-cardiovascular and anti-cancer effects in vitro and in vivo, epidemiological and clinical studies suggest an association with moderately reducing the risk of chronic diseases [39]. Chinese tea is generally divided into at least three categories on the basis of different production methods: nonfermented (green tea), semi-fermented (oolong tea), and fully fermented (black tea) [40].Among these, only black tea leaves are withered, rolled ,and fermented with bacteria [40].
Biologically active compounds containing anti-oxidative [41], anti-mutagenic [42], and anti-hypertriacylglycerolemia [43] properties have been observed in black tea leaves.
Kittaka-katsura et al. [44] demonstrated that the Japanese black tea leaf Batabata-cha contains approximately 0.5 mg of B12 per 100 g of dried tea leaves, which is bioavailable in mammals. In addition, they determined that B12 content of two types of Chinese black tea leaves [45] is, similar to that of Batabata-cha. However, B12 content in various types of Chinese black tea leaves and the identification of B12 compounds in Chinese black tea leaves as “true” B12 or inactive corrinoid compounds in humans remain to be established.
In Chapter IV, I described the characterization of B12 compounds from various Chinese fermented black tea leaves using TLC-E. coli 215 bioautography and LC/ESI-MS/MS.
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Materials and Methods
Materials
Authentic B12 was obtained from Sigma (St. Louis, Missouri, USA). Silica gel 60 TLC aluminum sheets were purchased from Merck (Darmstadt, Germany). All other reagents were high-grade and commercially available. Various types of Chinese black tea leaves (Pu’er, Ryubao, Fu, and Brick) were purchased from local markets in Japan (Fig.
14).
Extraction and assay of B12 from Chinese black tea leaf samples
Each sample (5 g) of dried Chinese black tea leaves was homogenized in a mixer (TML160; Tescom & Co., Ltd., Tokyo, Japan). A portion (2 g) of the homogenate was used as the test sample. B12 compounds of samples were extracted as described in Chapter II.
Tea from 3 g Ryubao leaves (sample H) with the highest B12 content among those tested was extracted for 5 min with 150 mL of boiling water. After cooling down to 40 ºC, the extract was used as a tea drink, and B12 was extracted from 50 mL of liquid using the above-mentioned method.
Bioautography of corrinoid compounds using B12-dependent E. coli 215
B12 extract (50 mL) prepared as described above was partially purified and concentrated using Sep-Pak Plus® C18 cartridge (Waters Corp., Milford, USA).
Bioautography of the B12 compounds was done according to the method of Tanioka et al.
[24].
36 Identification of B12 compounds by LC/ESI-MS/MS
Sample H (10 g) containing high levels of B12 was suspended in 500 mL of distilled water and homogenized in a mixer (TML 160). The homogenate was added to 50 mL of 0.57 mol/L acetic buffer (pH 4.5) with 0.05 g KCN and boiled for 30 min to extract B12
compounds. Extraction procedures were performed in a draught chamber (Dalton Co., Tokyo, Japan).The boiled suspension was centrifuged at 5,000 × g for 10 min. An aliquot (approximately 200 mL) of the supernatant was placed in Sep-pakVac 20cc (5 g) C18 cartridges (Waters Corp.) prewashed with 75% (v/v) ethanol and equilibrated with distilled water. The C18 cartridges were washed with 30 mL of distilled water and B12
compounds were eluted using 30 mL of 75% (v/v) ethanol. The remaining supernatant was treated in the same manner. Combined eluates were evaporated to dryness under reduced pressure, and the residual fraction was dissolved in 5 mL of distilled water and centrifuged at 10,000 × g for 10 min to remove any insoluble material.The supernatant fraction was loaded onto EASI-EXTRACT B12 Immunoaffinity Column (P80) [R-Biopharm AG, Darmstadt, Germany], and corrinoids were purified according to the manufacturer’s protocol. B12 compounds, pseudo-B12, and B12 were dissolved in 0.1%
Fig. 14. Types of Chinese black tea leaves. Pu’er tea (samples A-G), Ryubao tea (sample H), Fu tea (sample I), and Brick tea (sample J) leaves were used in this study.
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(v/v) acetic acid and filtered using a Nanosep MF centrifual device (0.4 μm, Pall Corp., Tokyo, Japan) to separate small particles. Aliquots (2 μL) of filtrate were analyzed using LCMS-IT-TOF coupled with an Ultra-Fast LC system (Shimadzu, Kyoto, Japan). Each purified corrinoid was injected into an InertSustain column (3 μm, 2.0 × 100 mm, GL Science, Tokyo, Japan) and equilibrated with 85% solvent A [0.1% (v/v) acetic acid)] and 15% solvent B (100% methanol) at 40 °C. Corrinoid compounds were eluted using a linear gradient of methanol (15% solvent B for 0 - 5 min, increasing the concentration from 15% to 90% solvent B for 5 - 11 min, followed by decreasing the concentration from 90% to 15% solvent B for 11 - 15 min) at a flow rate of 0.2 mL/min. ESI conditions were determined by injecting pseudo-B12 or B12 into the MS detector to ascertain the optimum parameters for detecting the parent B12 compound and daughter ions. ESI-MS was operated in the positive ion mode with argon as collision gas. Pseudo-B12 (m/z 672.777) and B12 (m/z 678.292) as [M + 2H] 2+ were confirmed by comparing the observed molecular ions and retention times molecular ions and retention times.
Results and Discussion
B12 contents in black tea leaves
B12 levels were assayed in 10 Chinese black tea leaves that are commercially available worldwide using the microbiological B12 assay method based on L. delbrueckii ATCC 7830 (Table 5). Traces (0.25 - 0.69 μg/100 g dry weight) of the corrected B12 were observed in Pu`er, Fu, and Brick tea leaves. However, Ryubao tea leaves (sample H) contained the highest B12 content (1.37 μg/100 g dry weight), which is similar to that previously reported [45]. To further clarify whether Ryubao tea leaves generally contain
38
high levels of B12, we determined the B12 content of the other Ryubao leaf samples. As shown in Table 6, the corrected B12 content of various Ryubao tea leaves varied (0.06 - 1.37 μg/100 g dry weight), and their mean value was calculated as approximately 0.69 μg of B12, which is only slightly higher than that for Pu`er tea leaves (approximately 0.49 μg/100 g dry weight). High levels (0.61 - 2.02 μg B12 equivalent/100 g dry weight) of the alkali-resistant factor were detected in all tested Chinese black tea leaves.
E. coli 215 Bioautography Analysis
B12 compounds identified in Chinese black tea leaf samples A-J were analyzed using the E. coli 215 bioautogram after separation using silica gel 60 TLC (Fig. 15). The Ryubao tea leaf extract (sample H) produced a single, clear spot with an Rf value identical
Vitamin B12content (μg/100 g dry weight) Apparent B12 Alkali-resistant
factor Corrected B12 Pu`er tea leaves
Sample A 1.94 1.32 0.62
Sample B 1.75 1.50 0.25
Sample C 2.55 1.86 0.69
Sample D 1.53 1.05 0.48
Sample E 2.35 1.77 0.58
Sample F 1.52 1.07 0.45
Sample G 2.42 2.02 0.40
Ryubao tea leaves
Sample H 2.94 1.57 1.37
Fu tea leaves
Sample I 1.87 1.30 0.57
Brick tea leaves
Sample J 0.74 0.61 0.13
Mean ±SD 1.96 ±0.63 1.41 ±0.43 0.55 ±0.33
Table 5. Vitamin B12 content of various types of Chinese black tea leaves.
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to that of authentic B12. Indistinct spots with the Rf value identical to that of authentic B12
were detected in samples C, D, E, G, and I. The remaining samples showed no spot because of their lower B12 contents.
Vitamin B12content (μg/100 g dry weight)
Apparent B12 Alkali-resistant
factor Corrected B12
Sample H 2.94 1.57 1.37
Sample K 1.57 1.23 0.34
Sample L 2.89 1.90 0.99
Sample M 1.68 1.62 0.06
Mean ±SD 2.27 ±0.75 1.58 ±0.27 0.69 ±0.60
1 2 A B C D E 1 2 F G H I J Table 6. Vitamin B12 content of various types of Ryubao tea leaves.
Fig. 15. E. coli 215 bioautogram analysis of B12 compounds detected in various black tea leaf samples.Authentic B12 (1), pseudo-B12 (2), and concentrated extracts of various black tea leaf samples A-J. Typical bioautograms from three independent experiments are presented.
40 LC/ESI-MS/MS analysis
To more precisely identify the corrinoid compounds present in Chinese black tea leaves, corrinoids were purified from the Ryubao tea leaf extract (sample H) containing high B12 content and identified using LC/ESI-MS/MS (Fig. 16). Authentic B12 was eluted as a peak with a retention time of 7.5 min. The mass spectrum of authentic B12 primarily comprised a doubly charged ion with m/z 678.2910 [M + 2H] 2+ (Fig. 16 A and B).
MS/MS spectra revealed a predominant monovalent ion with m/z 359.0994, which was largely attributable to the nucleotide moiety of B12 (Fig. 16 C). The corrinoid purified from the Ryubao tea leaf sample H was eluted as several ion peaks, indicating the presence of impurities. The mass spectrum of the main peak with m/z 687.2914 had a retention time of 7.5 min in the purified sample (Fig. 16 D and E). The MS/MS spectrum of the purified compound with a monovalent ion with m/z 359.0960 was identical to that of authentic B12 (Fig. 16 C and F). These results indicate that the Ryubao tea leaf sample H contained authentic B12 but not pseudo-B12 which is inactive in humans.
B12 content in the tea drink prepared from the Ryubao tea leaf sample H was 0.8 ng/100mL of black tea. Therefore, consumption of approximately 300 L of this tea would provide the recommended dietary allowance for adults (2.4 μg/day) [13], although ingestion of such large quantities of tea on a daily basis is not recommended. Notably, Kittaka-Katsura et al. [44] demonstrated that administration of the Japanese black tea drink (B12 content, approximately 2.0 ng/100mL) considerably improves B12 status in B12-deficient rats. Considering these earlier observations and our present findings, we propose that Ryubao tea leaves containing significantly levels of B12 can be utilized as a source of B12 for vegetarians.
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Fig. 16. LC/ESI-MS/MS chromatograms of authentic B12 and the B12 compounds purified from black tea leaf sample H. B12 compounds were analyzed using the LC/MS-IT-TOF system (Shimadzu).
Panels A and D show total ion chromatograms and those (m/z 678.2914) of authentic B12 and B12
compounds purified from sample H. Mass spectra of authentic B12 and purified B12 compounds at 7.5 min are shown in panels B and E, respectively (magnified spectrum range from m/z 678 to m/z 680 is shown as an insert in each panel). MS/MS spectra for the peak of authentic B12 at m/z 678.2910 and that of purified B12 compounds at m/z 678.2884 are shown in panels C and F, respectively.
42
Summary
I determined B12 content of Chinese black tea leaves using a microbiological assay based on L. delbrueckii ATCC 7830. Trace levels (0.25 - 0.69 μg/100 g dry weight) of B12 were detected in Pu’er, Fu, and Brick tea leaves. However, B12 content (0.06 - 1.37 μg/100 g dry weight) of Ryubao tea leaves significantly varied. To determine whether Chinese black tea leaves contain B12 or other corrinoid compounds that are inactive in humans, corrinoid compounds were purified from Ryubao tea by an immunoaffinity column chromatography and B12 was identified by LC/ESI-MS/MS. B12 content in the tea drink prepared from Ryubao tea leaves was very low (0.8 ng/100 mL). Our results indicate that Chinese black tea is usually not a good source of B12, although Ryubao tea leaves with the highest B12 content may be utilized as a source of this vitamin for vegetarians.
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